scholarly journals Inhibition of Oxidative Cross-linking between Engineered Cysteine Residues at Positions 332 in Predicted Transmembrane Segments (TM) 6 and 975 in Predicted TM12 of Human P-glycoprotein by Drug Substrates

1996 ◽  
Vol 271 (44) ◽  
pp. 27482-27487 ◽  
Author(s):  
Tip W. Loo ◽  
David M. Clarke
Keyword(s):  
Cross Linking ◽  
Cysteine Residues ◽  
Transmembrane Segments ◽  
P Glycoprotein

scholarly journals Correctors Promote Maturation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)-processing Mutants by Binding to the Protein

2007 ◽  
Vol 282 (46) ◽  
pp. 33247-33251 ◽  
Author(s):  
Ying Wang ◽  
Tip W. Loo ◽  
M. Claire Bartlett ◽  
David M. Clarke
Keyword(s):  
Cystic Fibrosis ◽  
Channel Blocker ◽  
Cross Linking ◽  
Transmembrane Conductance Regulator ◽  
Mature Protein ◽  
Transmembrane Conductance ◽  
P Glycoprotein ◽  
Δf508 Cftr ◽  
Cross Linker ◽  
Cystic Fibrosis Transmembrane

The most common cause of cystic fibrosis (CF) is defective folding of a cystic fibrosis transmembrane conductance regulator (CFTR) mutant lacking Phe508 (ΔF508). The ΔF508 protein appears to be trapped in a prefolded state with incomplete packing of the transmembrane (TM) segments, a defect that can be repaired by expression in the presence of correctors such as corr-4a, VRT-325, and VRT-532. To determine whether the mechanism of correctors involves direct interactions with CFTR, our approach was to test whether correctors blocked disulfide cross-linking between cysteines introduced into the two halves of a Cys-less CFTR. Although replacement of the 18 endogenous cysteines of CFTR with Ser or Ala yields a Cys-less mutant that does not mature at 37 °C, we found that maturation could be restored if Val510 was changed to Ala, Cys, Ser, Thr, Gly, Ala, or Asp. The V510D mutation also promoted maturation of ΔF508 CFTR. The Cys-less/V510A mutant was used for subsequent cross-linking analysis as it yielded relatively high levels of mature protein that was functional in iodide efflux assays. We tested for cross-linking between cysteines introduced into TM6 and TM7 of Cys-less CFTR/V510A because cross-linking between TM6 and TM7 of P-glycoprotein, the sister protein of CFTR, was inhibited with the corrector VRT-325. Cys-less CFTR/V510A mutant containing cysteines at I340C(TM6) and S877C(TM7) could be cross-linked with a homobifunctional cross-linker. Correctors and the CFTR channel blocker benzbromarone, but not P-glycoprotein substrates, inhibited cross-linking of mutant I340C(TM6)/S877C(TM7). These results suggest that corrector molecules such as corr-4a interact directly with CFTR.

scholarly journals Site-Directed Disulfide Cross-Linking Shows that Cleft Flexibility in the Periplasmic Domain Is Needed for the Multidrug Efflux Pump AcrB of Escherichia coli

2007 ◽  
Vol 189 (23) ◽  
pp. 8677-8684 ◽  
Author(s):  
Yumiko Takatsuka ◽  
Hiroshi Nikaido
Keyword(s):  
Escherichia Coli ◽  
Conformational Changes ◽  
Disulfide Bonds ◽  
Drug Transport ◽  
Site Directed Mutagenesis ◽  
Cross Linking ◽  
Cysteine Residues ◽  
Multidrug Efflux ◽  
Specific Chemical ◽  
Periplasmic Domain

ABSTRACT Escherichia coli AcrB is a multidrug efflux transporter that recognizes multiple toxic chemicals having diverse structures. Recent crystallographic studies of the asymmetric trimer of AcrB suggest that each protomer in the trimeric assembly goes through a cycle of conformational changes during drug export. However, biochemical evidence for these conformational changes has not been provided previously. In this study, we took advantage of the observation that the external large cleft in the periplasmic domain of AcrB appears to become closed in the crystal structure of one of the three protomers, and we carried out in vivo cross-linking between cysteine residues introduced by site-directed mutagenesis on both sides of the cleft, as well as at the interface between the periplasmic domains of the AcrB trimer. Double-cysteine mutants with mutations in the cleft or the interface were inactive. The possibility that this was due to the formation of disulfide bonds was suggested by the restoration of transport activity of the cleft mutants in a dsbA strain, which had diminished activity to form disulfide bonds in the periplasm. Furthermore, rapidly reacting, sulfhydryl-specific chemical cross-linkers, methanethiosulfonates, inactivated the AcrB transporter with double-cysteine residues in the cleft expressed in dsbA cells, and this inactivation could be observed within a few seconds after the addition of a cross-linker in real time by increased ethidium influx into the cells. These observations indicate that conformational changes, including the closure of the external cleft in the periplasmic domain, are required for drug transport by AcrB.

Cross-linking of S-nitrosothiolated AIEgen inside cancer cells to monitor NO release and reverse chemo-resistance

2021 ◽  
Author(s):  
Wen WU ◽  
Fan Ying ◽  
Luo Ting Rong ◽  
Yuchen Hu ◽  
Qingyu Zhang ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Multidrug Resistance ◽  
Cancer Cells ◽  
Cross Linking ◽  
Glycoprotein P ◽  
P Glycoprotein ◽  
No Release

Nitric oxide (NO)-releasing platforms have been demonstrated as promising approaches for the reversal of multidrug resistance (MDR) in cancer cells due to the suppression of P-glycoprotein (P-gp). However, the non-specific...

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